System and method for single molecule detection

ABSTRACT

A single molecule sensing or detecting device includes a first electrode and a second electrode separated from the first electrode by a gap. The first electrode and the second electrode have an opening formed therethrough. At least one of the first electrode and the second electrode is functionalized with a recognition molecule. The recognition molecule has an effective length L1 and is configured to selectively bind to a target molecule having an effective length L2. The size of the gap is configured to be greater than L2, but less than or equal to the sum of L1 and L2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/375,901, filed on Dec. 12, 2016, which claims priority to and thebenefits of U.S. Provisional Application No. 62/266,282, filed on Dec.11, 2015, the contents of each of which are incorporated herein byreference in their entireties.

GOVERNMENT RIGHTS

This invention was made with government support under R01 HG006323awarded by The National Institutes of Health. The government has certainrights in the invention.

ABSTRACT OF THE DISCLOSURE

The present disclosure presents systems, methods and devices fordetecting single molecules by direct electronic measurement as they binda cognate ligand. In some embodiments, high contrast signals areproduced with no labels and sample concentrations in the femtomolarrange.

BACKGROUND

Electron tunneling is, in principle, sensitive to the presence of amolecule in a tunnel gap formed between two closely spaced metalelectrodes (Zwolak and Di Ventra 2005). However, in practice, tunnelgaps are quite insensitive to molecules that may be trapped between theelectrodes because the inevitable hydrocarbon contamination of metalelectrodes outside of an ultrahigh vacuum clean environment makes for apoor contact between the electrodes and the molecules.

It has been shown that reproducible and characteristic electricalsignals can be obtained if molecules are chemically attached to eachelectrode forming a tunnel junction, by, for example, sulfur-metal bonds(Cui, Primak et al. 2001). Such permanent connections, however, do notmake for versatile detectors because the molecule that bridges the gapmust be modified at two sites with groups such as thiols. Pishrody etal. (Pishrody, Kunwar et al. 2004), proposed a solution in whichelectrode pairs were functionalized with molecules that did not, bythemselves bridge the gap, but rather, formed a bridged structure when atarget molecule became bound. This prior art is illustrated in FIG. 1.As shown, a first metal electrode 10 and a second metal electrode 12 areseparated by a dielectric layer 16 with the electrode gap exposed at theedge of the layered device. A first recognition molecule 14 a and asecond recognition molecule 14 b are chemically tethered to theelectrodes by reactive groups 18. The molecules 14 a and 14 b are chosenso as to bind a target molecule 20 in such a way as to form a bridgeacross the gap between the electrodes when 20 binds both 14 a and 14 b.For example 14 a and 14 b may be composed of DNA oligomers chosen sohave a sequence that, taken together, is complementary to a target DNAmolecule 20. However, the simple device of FIG. 1 cannot be used as asingle molecule detector, but rather, only as a system of a large numberof such devices functionalized with many pairs of recognition molecules.In this way, the presence of certain molecules in a sample could bedetermined upon the measurement of current from many binding events.

U.S. publication no. 2010/0084276 (Lindsay et al.) discloses a devicedesigned for sequencing polymers, such as DNA. In some embodiments ofthis prior art, as illustrated in FIG. 2, two closely spaced electrodes30, 31 are separated by dielectric layer 33. A nanopore 34 is thendrilled through the structure and the exposed electrodes functionalizedwith recognition molecules 35. The molecules bind to a target analyte 36at two separate sites. Thus, once an analyte molecule enters the pore,it brings together the recognition molecules to form a connected pathwayacross the gap. The approach of such embodiments differ from that ofPishrody at least because (a) the nanopore of Lindsay et al. permitsonly one analyte to enter at a time (e.g., so that a polymer may besequenced, as each chemical unit of the polymer enters the pore andgenerates a characteristic signal), and (b) the gap between theelectrodes 30 and 31 is sized such that that single molecule bindingevent generates a large current.

SUMMARY OF SOME OF THE EMBODIMENTS OF THE DISCLOSURE

It is an object of at least some of the embodiments of the presentdisclosure to provide a device that detects single molecule bindingevents by, for example, direct electronic detection of binding on only asingle ligand, e.g., such as an antibody.

It is another object of at least some of the embodiments of the presentdisclosure to provide a device with a large exposed junction areaconfigured for sensing low concentrations of samples rapidly. Forexample, in some embodiments, such junction areas correspond to junctiongaps of from 0.1 to 100 nm, with the lateral extent of the junctionsranging from 1 nm to 100 microns. Sample concentrations can be as low asone femtomolar, or even lower. A large junction area can be configuredto collect molecules from a large sample volume, so that the time formolecules to diffuse into the junction can be small. For example, for ajunction of a few microns in lateral extent, and a gap size of 4 nm,exposure to a concentration of 100 femtomoles results in generation ofsignals in about 10 s.

In some embodiments, a device for sensing molecules in solution isprovided which includes a first electrode and a second electrodeseparated from the first electrode by a gap. One or more of theelectrodes are functionalized with one or more recognition moleculeshaving an effective length L1 and configured to selectively bind to atarget molecule having an effective length L2. The gap is configured tobe greater than L2, but less than or equal to the total of L1 and L2.

In some embodiments, a method for sensing molecules in solution isprovided, which includes providing the device according to someembodiments of the disclosure (e.g., the device embodiment above),applying a voltage bias across the electrodes, providing a sample to thedevice, monitoring current over time to determine at least one of thefeatures thereof of a background and noise spikes, and determining,based on at least one of the background and noise spikes, determiningone or more of: the presence of the target molecule; and a number ofnon-target molecules adsorbed on the first electrode and/or on thesecond electrode.

In some embodiments, a device includes a first electrode and a secondelectrode separated from the first electrode by a gap. At least one ofthe first electrode and the second electrode is functionalized with arecognition molecule. The recognition molecule has an effective lengthL1 and is configured to selectively bind to a target molecule having aneffective length L2. The gap is configured to be greater than L2 inthickness, but less than or equal to the sum of L1 and L2.

In some embodiments, a method includes applying a voltage bias across afirst electrode and a second electrode of a device. The second electrodeis separated from the first electrode by a gap. At least one of thefirst electrode and the second electrode is functionalized with arecognition molecule that has an effective length L1 and is configuredto selectively bind to a target molecule having an effective length L2.The method also includes contacting the first electrode and the secondelectrode with a solution containing the target molecule in aconcentration from about 10 fM to about 10 pM. The method also includesmonitoring current generated between the first electrode and the secondelectrode over time. The method also includes determining one or moreof: based on a fluctuating portion of the current, the presence of thetarget molecule; and based on a background portion of the current, anumber of non-target molecules adsorbed on the first electrode and onthe second electrode.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a bridged electrode pair according to the prior art.

FIG. 2 illustrates a bridged electrode pair within a nanopore accordingto the prior art.

FIG. 3 illustrates a model of αvβ₃ integrin.

FIGS. 4a-b illustrate (a) Cyclic RGD peptide. (b) Binding site of RGDpeptide at the junction of the α and β subunits of integrin.

FIG. 5 illustrates (a) Scanning tunneling microscope experiment todemonstrate capture of αvβ₃ integrin with functionalization of just oneelectrode. (b) Typical current trace when an integrin is captured as theprobe is withdrawn.

FIG. 6 illustrates (a) histogram of current peak values for integrincapture (current is shown increasing negative to the left here). (b)Distribution of withdrawal distances to the peak signal (the startinggap, 0 on this plot, is 2.7 nm).

FIG. 7, illustrates a cross-sectional view of a tunnel junction edge ofa device as exposed by an opening, according to some embodiments of thepresent disclosure.

FIG. 8 illustrates current data recorded for two concentrations of α₄β₁integrin, a molecule not bound by the RGD peptide with a deviceaccording to some embodiments.

FIG. 9 illustrates current data from a device according to someembodiments, in buffer solution (a) and then after adding 1 pM (b), andthen 10 pM (c) of αvβ₃ integrin.

FIG. 10 illustrates control signals (a, buffer, b, α₄β₁integrin) for ananopore device according to some embodiments of the present disclosureinto which only a single molecule can be received.

FIG. 11 illustrates a signal obtained when 1 nM αvβ₃ integrin was placedin contact with the nanopore device according to some embodiments of thedisclosure.

FIG. 12 illustrates cyclic voltammetry of a palladium electrode of adevice according to some embodiments of the present disclosurefunctionalized with cyclic RGD peptide and exposed to a αvβ₃ integrinsolution. Scale is millivolts relative to a silver wire quasi reference.

FIG. 13 illustrates output of a device according to some embodimentswith a large (micron sized) junction length exposed to a 10 pM solutionof α₄β₁ integrin (lower trace) followed by a 10 femtomolar solution ofαvβ₃ integrin (upper trace).

DESCRIPTION OF THE PRESENT INVENTION IN ITS PREFERRED EMBODIMENT

A single molecule sensing or detecting device includes a first electrodeand a second electrode separated from the first electrode by a gap. Thefirst electrode and the second electrode have an opening formedtherethrough. At least one of the first electrode and the secondelectrode is functionalized with a recognition molecule. The recognitionmolecule has an effective length L1 and is configured to selectivelybind to a target molecule having an effective length L2. The size of thegap is configured to be greater than L2, but less than or equal to thesum of L1 and L2.

In some embodiments, the device further includes an insulating layerdisposed in the gap, wherein a thickness of the insulating layer is lessthan or equal to the sum of L1 and L2. In some embodiments, the size ofthe gap is at least twice the effective length L1. In some embodiments,the size of the gap is equal to the sum of L1 and L2. In someembodiments, the size of the gap is between about 2 nm to about 15 nm.In some embodiments, the size of the gap is between about 2 nm to about10 nm. In some embodiments, the size of the gap is between about 5 nm toabout 15 nm. In some embodiments, the recognition molecule includes anysuitable peptide such as, for example, a cyclic RGD peptide. In someembodiments, the size of the opening is between 0.1 nm and 100 micronsin a linear dimension.

In some embodiments, the first electrode and/or the second electrode areconfigured to generate a current upon binding of the target molecule.and the current includes a fluctuating portion and/or a backgroundportion. In some embodiments, the background portion of the current isbased on a number of non-target molecules adsorbed on the firstelectrode and/or on the second electrode. In some embodiments, thefluctuating portion is based on a concentration of the target moleculein a solution containing the target molecule, the solution in contactwith the first electrode and the second electrode, and the concentrationof the target molecule in the solution is from about 10 fM to about 1μM.

In some embodiments, a method for sensing or detecting a target moleculeincludes applying a voltage bias across a first electrode and a secondelectrode of a molecular sensing or detecting device. The firstelectrode and second electrode collectively have an opening formedtherethrough. The second electrode separated from the first electrode bya gap, and at least one of the first electrode and the second electrodeis functionalized with a recognition molecule. The recognition moleculeincludes an effective length L1 and is configured to selectively bind toa target molecule having an effective length L2. The method alsoincludes contacting the first electrode and the second electrode with asolution containing the target molecule in a concentration from about 10fM to about 1 μM. The method also includes monitoring current generatedbetween the first electrode and the second electrode over time. Themethod also includes determining one or more of: the presence of thetarget molecule; and a number of non-target molecules adsorbed on thefirst electrode and/or on the second electrode.

In some embodiments, determining the presence of the target molecule isbased on a fluctuating portion of the current. In some embodiments,determining a number of non-target molecules adsorbed on the firstelectrode and/or on the second electrode is based on a backgroundportion of the current. In some embodiments, the device further includesan insulating layer disposed in the gap, and a thickness of theinsulating layer is less than or equal to the sum of L1 and L2. In someembodiments, the gap is at least twice the effective length L1 inthickness. In some embodiments, the size of the gap is equal to the sumof L1 and L2. In some embodiments, the size of the gap is between about2 nm to about 15 nm. In some embodiments, the size of the gap is betweenabout 2 nm to about 10 nm. In some embodiments, the size of the gap isbetween about 5 nm to about 15 nm. In some embodiments, the recognitionmolecule includes a peptide. In some embodiments, the peptide is acyclic RGD peptide.

It is commonly assumed that proteins are excellent insulators. Directmeasurements of the conductance of small peptides (i.e., short proteinfragments) in their linear form shows that current decays very rapidlywith an increase in the length (i.e., number of amino acid residues) ofthe peptide (Xiao, Xu et al. 2004). However, scanning-tunnelingmicroscope studies of electron-transfer proteins (Ulstrup 1979, Artes,Diez-Perez et al. 2012), can show remarkably large conductance values.While these values are impossible to reconcile with the short electronicdecay lengths measured in peptides, it has recently been suggested thatmany proteins, in their three dimensional, folded form, are poised in acritical state between being a bulk conductor (metal-like) and aninsulator, such that local fluctuations can drive proteins into statesthat are transiently conductive (Vattay, Salahub et al. 2015).Accordingly, some embodiments of the present disclosure are disclosedwhich enable proteins to form highly conductive bridges across gapsbetween electrodes that are much larger than could possibly supportelectron tunneling currents. Even with the most favorable electronicproperties of a molecule in a tunnel junction, tunnel conductances dropbelow femtoseimens for distances of 3 to 4 nm. Such large gaps provide,in at least some embodiments, a large current signal, even when thetarget protein is bound to only one electrode by a recognition reagent,with currents corresponding to nanoseimens of conductance.

EXAMPLES

To illustrate the process we use the example of αvβ₃ integrin, whichcomprises two subunits (the α and β chains) that meet at the apex ofpyramidal shape that is about (in some embodiments) 9 nm high (FIG. 3).This protein is strongly bound by a cyclic RGB peptide (FIG. 4a ) at aunique site near the apex of the pyramidal shape (FIG. 4b ). In FIG. 4b, 41 is the junction between the α and β chains and 40 is the cyclic RGDpeptide (Choi, Kim et al. 2010). The peptide is relatively small beingabout 1 nm across its widest folded dimension.

Accordingly, in some embodiments, functionalizing just one of a pair ofelectrodes generates a unique electrical recognition signal for acorresponding molecule(s). To do this, a scanning tunneling microscope(STM) was used (see STM, FIG. 5a ), where a gold probe 51 wasfunctionalized with the RGD peptide 53 via the chemical interactionbetween the cysteine residue and the gold. The probe was positioned at aset point bias (V) and current (I) such that the apex of the probe washeld approximately 2.7 nm above a bare gold substrate, 52. As the probewas pulled away an extra distance ΔZ from the surface, a decayingcurrent could be observed during some of the experiments (e.g., feature57 in FIG. 5b ). However, in the absence of the αvβ₃ integrin, no otherfeatures were observed, even if fairly high concentrations (e.g., 100nM) of a protein such as BSA were added to the solution in the STM. Once100 nM αvβ₃ integrin 56 was added to the solution, a new featureappeared as a current peak away from the origin 58 in FIG. 5 b.

A statistical analysis of the distribution of features in terms of thepeak current (FIG. 6a ) and distance above approximately 2.7 nm forwhich a peak occurs (ΔZ in FIG. 6b ) shows that signals of many tens ofpicoamps are generated at distances of about 3 nm to about 6 nm overall(2.7 nm +ΔZ). In contrast to conventional tunneling signals, thesesignals peak when the probe is some distance away from the surface,signifying that the probe has captured a conductive particle.Importantly, no such features were seen in the absence of the αvβ₃integrin, or in the presence of a protein (BSA) that does not bind thecyclic RGD peptide.

FIG. 7 illustrates a cross-sectional view of a tunnel junction edge(i.e., the edge of an opening) of the device according to someembodiments of the present disclosure which includes a first metalelectrode 71 (e.g., palladium, gold and/or platinum) onto which isdeposited a layer of an insulating dielectric such as alumina 72 (forexample). A second metal electrode 73 is then deposited, typically usingone of the metals used for the first electrode. In some embodiments (notshown), the first electrode 71 and the second electrode 73 A cut is thenmade in the structure to expose the edge of these layers (fabrication ofthis type of device is described in detail in co-pending, publishedWO2015/161119, and also in Pang, Ashcroft et al. 2014) to form theopening/nanopore 78, shown here as a plane adjacent to the elctrodes 71,73. Accordingly, the first electrode 71 and the second electrode 73 canhave an opening formed therethrough such as, for example, a nanopore.Said another way, the first electrode 71 and the second electrode 73 canbe said to be arranged within or adjacent to an opening, or have ananopore formed therethrough.

In some such embodiments (of those illustrated in, e.g., FIG. 7), it isa particular feature of such embodiments in the use of, depending uponthe embodiments, either or both of (a) recognition molecules that bind atarget at only one site, so that the geometric constraints of forming achemical bridge do not apply, and (b) a choice of dimensions that givesa very high signal-to-background ratio. Specifically, for example, theinsulating layer 72 is deposited with a thickness d that is chosen to begreater than the longest linear dimension of the recognition molecules74 (L₁, referred to as its effective length). However, according to someembodiments, it is chosen to be less than the combined overall length ofthe largest dimension of a target molecule 75 (L₂, also referred to asits effective length) bound to the recognition molecule 74 (L₁+L₂). Inthe case of the integrin-RGD pair (FIGS. 3 and 4) the RGD molecule isabout 1 nm at its longest, while the integrin is 9 nm, for a total ofabout 10 nm. This is a distance over which electron tunneling currentsgenerally do not flow because the tunneling probability would beinfinitesimal. However, a large particle that fluctuates into a highlyconductive state could be used to mediate current flow (Vattay, Salahubet al. 2015). In some embodiments, the gap need only be madesubstantially larger than twice the largest dimension of the recognitionmolecules (i.e., >2L₁). Thus, for example, if a recognition molecule is1 nm at its longest dimension, then the gap is configured to be greaterthan 2 nm, preferably by about 10%, to accommodate variations in thejunction geometry (larger than 2.2 nm in this example). In someembodiments, a gap size can be from the noted minimum up to the size ofone recognition molecule plus the size of the target protein. Forexample, if the largest dimension of the protein is 9 nm, then the gapin this case can be as big 9 nm plus the size of one of the recognitionmolecules (1 nm in this example), thus, a gap of 10 nm. In experiments,devices functionalized with the cyclic RGD peptide and fabricated with agap d of 3.5 to 4 nm show no background current, which continues to bethe case even when the junctions are exposed to a homologous protein(α₄β₁ integrin) that does not bind the RGD peptide. FIG. 8 showscurrent-vs time traces for devices in contact with 10 pM (a) and 100 pM(b) solutions of α₄β₁ integrin.

However, when the junctions are exposed to the target protein (α_(v)β₃integrin) signals appear immediately. FIG. 9 shows (a) the signal in 1mM phosphate buffer (pH 7.0) just before the addition of a 1 pM solutionof α_(v)β₃ integrin in the same buffer solution (b). A clear signal isimmediately generated which includes two (2) features as marked: abackground current (of about 0.5 nA in this case) and noise spikes of0.5 to 1 nA superimposed on top. On increasing the concentration ofα_(v)β₃ integrin to 10 pM, the background current increases by nearly anorder of magnitude (while the fluctuations remain generally constant).

Accordingly, in some embodiments, the background signal corresponds tothe number of molecules adsorbed on the electrodes. This can besubstantiated by collecting signals from a device small enough to allowonly one integrin molecule to be trapped. In such a device, experimentswere performed where the electrode edges were exposed by drilling ananopore of approximately 12 nm diameter through the junction device.The electrodes were functionalized again with the cyclic RGD peptide.FIG. 10 shows that in phosphate buffer (a) or in the presence of a 1 nMsolution of the non-binding control (α₄β₁ integrin) no signals aregenerated. However, when 1 nM αvβ₃ integrin is added a signal isgenerated (FIG. 11). Note that even though the concentration of theprotein is 100× that used to generate the signals shown in FIG. 9b ,there is essentially no background current, only the fluctuating currentcomponent (of about 0.2 nA in this case). This is because there is roomfor only one molecule at a time in the device, and this confirms that,in some embodiments, the background current arises from adsorption ofmany molecules.

Stable operation of the device requires control of the operatingpotential as described for similar devices in PCT publication no.WO2015/130781, entitled, “Methods, Apparatuses and Systems forStabilizing Nano-Electronic Devices in Contact with Solutions”, theentire disclosure of which is herein incorporated by reference. FIG. 12shows cyclic voltamograms taken with an RGD functionalized palladiumelectrode in the presence of a solution of αvβ₃ integrin. As shown,Faradaic current begins to rise above about 400 mV (with respect to asilver wire quasi reference electrode). Since, this is the upper limitof the bias applied to the across the electrode gap, the device operatesstably if one electrode is connected to a silver wire (or Ag/AgCl)reference and the other electrode is kept below +400 mV with respect tothe reference.

In experiments, the concentration used to obtain signals with the singlemolecule capture device had to be quite high (i.e., nanomolar or higher)in order for the probability of capturing a single molecule in areasonable time to be significant. In some embodiments, this probabilityis proportional to the volume from which molecules can be captured in areasonable time. For example, if the molecules diffuse freely with adiffusion constant D (e.g., about 10⁻¹¹m²/s), then the volume from whichmolecules can be collected in a time t, over a linear junction length L,is given approximately by πr²L where r²=Dt. Taking t=60 s and L=36 nm(approximately the length of the junction around the edge of a 12 nmdiameter pore), about 40 molecules would be present at 1 nMconcentration in the resulting volume of 6.5×10⁻¹⁷ m³ (=6.5×10⁻¹⁴liters). Referring to FIG. 7, if the junction length, X, is greatlyincreased (over the value of L given for the perimeter of a nanoporeearlier, L=36 nm in the example given) then correspondingly, thesensitivity of the device will also increase. Thus, for a device withX=10 μm, the capture volume in 1 minute capture time becomes 10⁻¹⁴ m³ oralmost 100× greater. Thus, signals are readily obtained at 1 pMconcentrations as shown in FIG. 9b . In fact, upon the solution beingflowed over the device, the effective capture length is orders ofmagnitude greater. For the cyclic RGD peptide, capturing αvβ₃ integrin,the binding process appears to be almost irreversible, so essentiallyall of the molecules within a capture radius can be swept up. Thus, ifabout a linear cm of fluid is flowed past a junction slowly enough thateach volume of length equal to the junction length spends about a minuteover the junction, then concentrations as small as a femtomole willyield a signal.

FIG. 13 shows an experiment in which a 10 pM solution of α₄β₁ integrinwas flowed over large (X=0.1 μm junction) for several minutes with nosignal being produced. When 10 fM of αvβ₃ integrin was introduced, alarge signal appeared after a few minutes, which substantially exceedsthe sensitivity estimated above (where much longer exposure times wouldbe required for the even larger (X=10 μm) junction geometry.

One of skill in the art recognizes that the specific dimensions givenhere are exemplary only. For example, a much larger gap (e.g., 5 to 15nm), can be used if the recognition molecules (cognate ligands) are fullsized antibodies (e.g., about 10 nm in extent), so the gap size (d inFIG. 7) would be, for example, 20 nm. An alternative to antibodies couldbe single-domain antibodies such as those produced by Abcore Inc. (forexample). Such single-domain antibodies include molecular weights of 50kD and linear dimensions of around 2.5 nm, so gaps of 5 nm would beappropriate.

Any and all references to publications or other documents, including butnot limited to, patents, patent applications, articles, webpages, books,etc., presented anywhere in the present application, are hereinincorporated by reference in their entirety.

As noted elsewhere, the disclosed embodiments have been presented forillustrative purposes only and are not limiting. Other embodiments arepossible and are covered by the disclosure, which will be apparent fromthe teachings contained herein. Thus, the breadth and scope of thedisclosure should not be limited by any of the above-describedembodiments but should be defined only in accordance with claimssupported by the present disclosure and their equivalents. Moreover,embodiments of the subject disclosure may include methods, compositions,systems and apparatuses/devices which may further include any and allelements from any other disclosed methods, compositions, systems, anddevices, including any and all elements corresponding to detecting oneor more target molecules (e.g., DNA, proteins, and/or componentsthereof). In other words, elements from one or another disclosedembodiments may be interchangeable with elements from other disclosedembodiments. Moreover, some further embodiments may be realized bycombining one and/or another feature disclosed herein with methods,compositions, systems and devices, and one or more features thereof,disclosed in materials incorporated by reference. In addition, one ormore features/elements of disclosed embodiments may be removed and stillresult in patentable subject matter (and thus, resulting in yet moreembodiments of the subject disclosure). Furthermore, some embodimentscorrespond to methods, compositions, systems, and devices whichspecifically lack one and/or another element, structure, and/or steps(as applicable), as compared to teachings of the prior art, andtherefore represent patentable subject matter and are distinguishabletherefrom (i.e. claims directed to such embodiments may contain negativelimitations to note the lack of one or more features prior artteachings).

Also, while some of the embodiments disclosed are directed to detectionof a protein molecule, within the scope of some of the embodiments ofthe disclosure is the ability to detect other types of molecules.

When describing the molecular detecting methods, systems and devices,terms such as linked, bound, connect, attach, interact, and so forthshould be understood as referring to linkages that result in the joiningof the elements being referred to, whether such joining is permanent orpotentially reversible. These terms should not be read as requiring aspecific bond type except as expressly stated.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of” or, when used inthe claims, “consisting of,” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of” “only one of” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

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What is claimed is:
 1. A sensing device for sensing protein fluctuationscomprising: a first electrode; a second electrode separated from thefirst electrode by a gap, a first recognition molecule having a firstend and a second end, the first end being connected to the firstelectrode and the second end being connected to a protein at a firstpoint; a second recognition molecule having a first end and a secondend, the first end being connected to the second electrode and thesecond end being connected to the protein at a second point; a means forapplying a voltage bias; and a detector configured to detectfluctuations in current.
 2. The sensing device of claim 1, wherein thefirst and the second recognition molecules are identical.
 3. The deviceof claim 1, wherein the size of the gap is between about 2 nm to about15 nm.
 4. The device of claim 1, wherein the size of the gap is betweenabout 2 nm to about 10 nm.
 5. The device of claim 1, wherein the size ofthe gap is between about 5 nm to about 15 nm.
 6. The device of claim 1,wherein the first and second recognition molecules comprise a peptide.7. A method for detecting protein fluctuations, the method comprising:applying a voltage bias across a first electrode to a second electrodeof a sensing device, the sensing device comprising: the first electrode;the second electrode separated from the first electrode by a gap, afirst recognition molecule having a first end and a second end, thefirst end being connected to the first electrode and the second endbeing connected to a protein at a first point; a second recognitionmolecule having a first end and a second end, the first end beingconnected to the second electrode and the second end being connected tothe protein at a second point; monitoring current generated between thefirst and second electrode over time; wherein protein fluctuation isdetected if the current generated fluctuates.
 8. The method of claim 7,wherein the first and the second recognition molecules are identical. 9.The method of claim 7, wherein the first and the second recognitionmolecules are identical.
 10. The method of claim 7, wherein the size ofthe gap is between about 2 nm to about 15 nm.
 11. The method of claim 7,wherein the size of the gap is between about 2 nm to about 10 nm. 12.The method of claim 7, wherein the size of the gap is between about 5 nmto about 15 nm.
 13. The method of claim 7, wherein the first and secondrecognition molecules comprise a peptide.